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Optimization of the Small Scale Expression of the Mutant Hen Egg White Lysozyme, H15S

Amoyaw, Charles Duah
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1587473897537747

Abstract Details

2020, Master of Science in Chemistry, Youngstown State University, Department of Chemistry.
Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules and radicals mainly produced from the partial reduction of molecular oxygen. ROS have been associated with aging and several diseases such as atherosclerosis and cancer. Metal-catalyzed oxidation (MCO) systems are systems that produce free radicals using transition metal ions such as copper or iron and hydrogen peroxide. As such, MCO may cause oxidation of proteins. To study the correlation between protein structure and oxidative damage of proteins by MCOs, different mutants of hen egg white lysozyme (HEWL) have been developed. This research focuses on the optimization of the expression of the mutant HEWL H15S. The Pichia pastoris expression system was adapted for the expression of HEWL H15S. The P. pastoris X-33-pPICZαA-hewlH15S strain was subjected to different growth conditions in a glycerol and methanol buffered media under conditions for small scale expression. Both intracellular and extracellular protein expression were analyzed for enzyme activity. Increasing glycerol concentration from 0.5% to 1% did not show significant increase in yeast growth resulting in low protein concentration and enzyme activity at 28 °C. Also, protein expression at three different methanol concentrations at 28 °C: 0.5% (v/v), 1% (v/v), and 2% (v/v) showed an increase in enzyme activity but only small changes in total protein concentration. The addition of calcium chloride showed a significant effect on the expression of H15S to about 1mg/mL compared to the other conditions without CaCl2. Lysing of the cells grown at 28 °C for intracellular analysis by the Bradford assay showed a significant band of protein corresponding to the size of the H15S mutant. A lower temperature of 22 °C at different growth and expression conditions measured high protein concentration and an increase in enzyme activity for extracellular expression. Intracellular analysis on protein expression at 22 °C measured no lysozyme activity.
Michael Serra, PhD (Advisor)
Nina Stourman, PhD (Committee Member)
John Jackson, PhD (Committee Member)
86 p.
Biochemistry; Biology; Biomedical Research; Chemistry; Microbiology
Site specific oxidation; oxygen species; metal catalyzed oxidation systems; MCO systems; HEWL; lysozyme; Pichia pastoris; reactive oxygen species; H15S; Mutant Hen Egg White Lysozyme; oxidative damage; protein oxidation

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Citations

  • Amoyaw, C. D. (2020). Optimization of the Small Scale Expression of the Mutant Hen Egg White Lysozyme, H15S [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1587473897537747

    APA Style (7th edition)

  • Amoyaw, Charles. Optimization of the Small Scale Expression of the Mutant Hen Egg White Lysozyme, H15S . 2020. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1587473897537747.

    MLA Style (8th edition)

  • Amoyaw, Charles. "Optimization of the Small Scale Expression of the Mutant Hen Egg White Lysozyme, H15S ." Master's thesis, Youngstown State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1587473897537747

    Chicago Manual of Style (17th edition)

ysu1587473897537747
323
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